Effects of alloferon and its analogues on reproduction and development of the Tenebrio molitor beetle.

As the most numerous group of animals on Earth, insects are found in almost every ecosystem. Their useful role in the environment is priceless; however, for humans, their presence may be considered negative or even harmful. For years, people have been trying to control the number of pests by using synthetic insecticides, which eventually causes an increased level of resistance to applied compounds. The effects of synthetic insecticides have encouraged researchers to search for alternatives and thus develop safe compounds with high specificity. Using knowledge about the physiology of insects and the functionality of compounds of insect origin, a new class of bioinsecticides called peptidomimetics, which are appropriately modified insect analogues, was created. One promising compound that might be successfully modified is the thirteen amino acid peptide alloferon (HGVSGHGQHGVHG), which is obtained from the hemolymph of the blue blowfly Calliphora vicinia. Our research aimed to understand the physiological properties of alloferon and the activity of its peptidomimetics, which will provide the possibility of using alloferon or its analogues in the pharmaceutical industry, as a drug or adjuvant, or in agriculture as a bioinsecticide. We used alloferon and its three peptidomimetics, which are conjugates of the native peptide with three unsaturated fatty acids with various chain lengths: caprylic, myristic, and palmitic. We tested their effects on the morphology and activity of the reproductive system and the embryogenesis of the Tenebrio molitor beetle. We found that the tested compounds influenced the growth and maturation of ovaries and the expression level of the vitellogenin gene. The tested compounds also influenced the process of egg laying, embryogenesis, and offspring hatching, showing that alloferon might be a good peptide for the synthesis of effective bioinsecticides or biopharmaceuticals.

Effect of paprika extracts on growth performance, haemolymph chemistry, intestinal microbiota and antioxidant enzyme activities of white-leg shrimp (Litopenaeus vannamei).

This study aims to evaluate the effects of paprika extract on the survival rate, growth performance and stimulation of the innate immune system of Litopenaeus vannamei. In this experiment, 240 healthy shrimp (3.22 ± 0.12 g) were randomly divided into four groups. The shrimp were fed diets with different concentrations of paprika oil extracts (0%, 0.5%, 1% and 2%) for 8 weeks. The results showed that growth performance, urea, uric acid, creatinine, cholesterol levels, aspartate aminotransferase and alkaline phosphatase activities were not significantly affected by adding paprika extract to the shrimp diet (p > 0.05). Diets containing 1% and 0.5% paprika extract showed the highest levels of total protein and triglyceride, respectively (p < 0.05). There was a significant decrease in haemolymph glucose concentration in shrimp-fed diets containing 1% and 2% paprika extract (p < 0.05). Moreover, a diet containing 0.5% paprika extract resulted in the highest levels of total heamocyte count, hyaline cells and large-granular cells in shrimp (p < 0.05). Higher catalase and superoxide dismutase activities were also exhibited in the paprika groups (p < 0.05). Vibrio sp. bacteria were not significantly reduced by paprika extract in the intestines of L. vannamei (p > 0.05). A significant decrease in heterotrophic bacteria was observed with increasing extract concentrations (p < 0.05). The shrimp culture industry can utilize paprika extract as a cost-effective, efficient and environmentally friendly immune stimulant at a concentration of 0.5%.

Z. morio Hemolymph Relieves E. coli-Induced Mastitis by Inhibiting Inflammatory Response and Repairing the Blood-Milk Barrier.

Escherichia coli (E. coli) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio (Z. morio) hemolymph against mastitis pathogens. In this study, we established E. coli-induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli-induced mastitis in vivo and in vitro. In mice with E. coli, Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1ß, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4+ T/CD8+ T, and increased the production of IL-2 triggered by E. coli. Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli-induced mastitis. In E. coli-stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli-induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli-induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli-induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

Systemic muscle wasting and coordinated tumour response drive tumourigenesis.

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. Here, we use a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. We demonstrate coordinate induction of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. We identify Indole-3-propionic acid as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network.

Osmotic and ionic regulation, and modulation by protein kinases, FXYD2 peptide and ATP of gill (Na+, K+)-ATPase activity, in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae).

We analyzed the modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+-calmodulin-dependent kinase, of gill (Na+, K+)-ATPase activity in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. Osmotic and ionic regulatory ability and gill (Na+, K+)-ATPase activity also were evaluated. (Na+, K+)-ATPase activity is stimulated by exogenous pig kidney FXYD2 peptide, while phosphorylation by endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase inhibits activity. Stimulation by FXYD2 and inhibition by protein kinase C and Ca2+/calmodulin-dependent kinase are salinity-dependent. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. At low salinities, the (Na+, K+)-ATPase exhibited a single, low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18‰S, a second, cooperative, high affinity ATP-binding site appeared, corresponding to 10-20% of total (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35‰S. Cl- was well hyper-/hypo-regulated although Na+ much less so, becoming isonatremic at elevated salinity. (Na+, K+)-ATPase activity was greatest in isosmotic crabs (26‰S), decreasing notably at 35‰S and also diminishing progressively from 18to 2‰S. Hyper-osmoregulation in U. cordatus showed little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of enzymatic and osmoregulatory adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.

Wastewater bioremediation by mangrove ecosystems impacts crab ecophysiology: In-situ caging experiment.

Mangroves are tidal wetlands that are often under strong anthropogenic pressures, despite the numerous ecosystem services they provide. Pollution from urban runoffs is one such threats, yet some mangroves are used as a bioremediation tool for wastewater (WW) treatment. This practice can impact mangrove crabs, which are key engineer species of the ecosystem. Using an experimental area with controlled WW releases, this study aimed to determine from an ecological and ecotoxicological perspective, the effects of WW on the red mangrove crab Neosarmatium africanum. Burrow density and salinity levels (used as a proxy of WW dispersion) were recorded, and a 3-week caging experiment was performed. Hemolymph osmolality, gill Na+/K+-ATPase (NKA) activity and gill redox balance were assessed in anterior and posterior gills of N. africanum. Burrow density decreased according to salinity decreases around the discharged area. Crabs from the impacted area had a lower osmoregulatory capacity despite gill NKA activity remaining undisturbed. The decrease of the superoxide dismutase activity indicates changes in redox metabolism. However, both catalase activity and oxidative damage remained unchanged in both areas but were higher in posterior gills. These results indicate that WW release may induce osmoregulatory and redox imbalances, potentially explaining the decrease in crab density. Based on these results we conclude that WW release should be carefully monitored as crabs are key players involved in the bioremediation process.

Effect of fluoranthene on antioxidative defense in different tissues of Lymantria dispar and Euproctis chrysorrhoea larvae.

This study examined the effect of long-term exposure to environmentally relevant concentrations of dietary fluoranthene (6.7 and 67 ng / g dry food weight) on defense mechanisms of the polyphagous forest insects Lymantria dispar L. and Euproctis chrysorrhoea L. The activities and expression of isoforms of superoxide dismutase (SOD) and catalase (CAT), the activities of glutathione S-transferase (GST) and glutathione reductase (GR), and total glutathione content (GSH) were determined in the whole midgut and midgut tissue, while SOD and CAT activities were assessed in hemolymph of the larvae. The results showed significant changes of enzyme activities, with more pronounced responses in larval midgut tissues, and between-species differences in patterns of response. Significantly increased activity of SOD was recorded in the whole midgut and midgut tissue of L. dispar larvae, as well as in midgut tissue of E. chrysorrhoea larvae. Fluoranthene increased CAT activity in midgut tissue of L. dispar larvae, and in the whole midgut and midgut tissue of E. chrysorrhoea larvae. Different expression patterns were detected for enzyme isoforms in tissues of larvae exposed to dietary fluoranthene. Total GSH content and GST activity increased in E. chrysorrhoea larval midgut tissue. Significantly decreased SOD activity in hemolymph of L. dispar larvae, and opposite changes in CAT activity were recorded in the hemolymph of larvae of two insect species. The tissue-specific responses of enzymes to dietary fluoranthene, recorded in each species, enabled the larvae to overcome the pollutant induced oxidative stress, and suggest further assessment of their possible use as early-warning signals of environmental pollution.

Biomphalaria alexandrina: a model organism for assessing the endocrine disrupting effect of 17ß-estradiol.

A wide range of endocrine disruptor compounds are routinely discharged to the ecosystem. Water contaminated with these compounds has a potential effect on the reproductive physiology of aquatic organisms as well as humans. In the present study, we tested the effect of the steroid estrogen, 17ß-estradiol, on Biomphalaria alexandrina, a snail species that is widely distributed in Egypt and that acts as an intermediate host for the human blood fluke, Schistosoma mansoni. The effects of exposure to 0.3 mg/L and 1 mg/L 17ß-estradiol on fecundity (MX) and reproductive rate (R0) of B. alexandrina were recorded. In addition, levels of steroid sex hormones and antioxidants in the hemolymph and ovotestis (OT) of exposed snails were measured. Histopathological changes in the OT of B. alexandrina were also investigated. Exposure to 0.3 mg/L and 1 mg/L 17ß-estradiol caused a significant increase in the number of egg masses per snail after 3 weeks and 1 week of exposure for the two tested concentrations compared with unexposed controls. An increase in the levels of progesterone hormone was recorded in the hemolymph of exposed snails in comparison with unexposed controls. Additionally, levels of the antioxidant enzyme glutathione (GSH) were increased in the hemolymph and OT tissues of snails after 2 and 4 weeks of exposure. Histopathological sections in the OT revealed an increase in the oocyte and a decrease in the sperm densities after 2 weeks and this effect was restored to normal conditions after 4 weeks of exposure to both tested concentrations. The current results indicate that B. alexandrina is sensitive to 17ß-estradiol and can therefore be used as bioindicator and model organism for the assessment of water pollution with endocrine disruptor compounds.

Sensitivity to near-future CO2 conditions in marine crabs depends on their compensatory capacities for salinity change.

Marine crabs inhabit shallow coastal/estuarine habitats particularly sensitive to climate change, and yet we know very little about the diversity of their responses to environmental change. We report the effects of a rarely studied, but increasingly prevalent, combination of environmental factors, that of near-future pCO2 (~1000 µatm) and a physiologically relevant 20% reduction in salinity. We focused on two crab species with differing abilities to cope with natural salinity change, and revealed via physiological and molecular studies that salinity had an overriding effect on ion exchange in the osmoregulating shore crab, Carcinus maenas. This species was unaffected by elevated CO2, and was able to hyper-osmoregulate and maintain haemolymph pH homeostasis for at least one year. By contrast, the commercially important edible crab, Cancer pagurus, an osmoconformer, had limited ion-transporting capacities, which were unresponsive to dilute seawater. Elevated CO2 disrupted haemolymph pH homeostasis, but there was some respite in dilute seawater due to a salinity-induced metabolic alkalosis (increase in HCO3- at constant pCO2). Ultimately, Cancer pagurus was poorly equipped to compensate for change, and exposures were limited to 9 months. Failure to understand the full spectrum of species-related vulnerabilities could lead to erroneous predictions of the impacts of a changing marine climate.

Effects of atrazine on vitellogenesis, steroid levels and lipid peroxidation, in female red swamp crayfish Procambarus clarkii.

Atrazine, a widely use herbicide, has been classified as a potential endocrine disruptor, especially for freshwater species. In this study, we tested the hypothesis that atrazine can affect reproduction in crayfish through dysregulation of vitellogenin expression and hormone synthesis. Adult female crayfish (Procambarus clarkii) were exposed during one month to atrazine at concentrations of either 1 or 5 mg/L. At the end of the exposure, ovaries, hepatopancreas, and hemolymph samples were harvested for analysis of vitellogenin expression and steroid hormone levels. Ovarian tissue was also sampled for both biochemical and histological analyses. Our results show that atrazine-exposed crayfish had a lower expression of vitellogenin in the ovary and hepatopancreas, as well as smaller oocytes, and reduced vitellogenin content in the ovary. Despite these effects, circulating levels of estradiol increased in females exposed to 5 mg/L of atrazine, showing that the inhibiting effect of atrazine on vitellogenin production was not related to a lower secretion of sexual steroids. Instead, some early stimulating effects of estradiol on vitellogenesis could have occurred, particularly in the hepatopancreas. On the other hand, atrazine caused a higher metabolic effort, in terms of lactate production, presumably triggered to provide the energy needed to face the unspecific stress produced by the herbicide. Lipid peroxidation was not affected by atrazine, but glutathione levels were significantly increased.

Effects of food contaminated with cadmium and copper on hemocytes of Steatoda grossa (Araneae: Theridiidae).

The aim of this study was to evaluate the metabolic condition of Steatoda grossa (Theridiidae) spider, from their hemocytes, after a short-term (four-week) exposure to cadmium and copper in sublethal doses by administering them into the body of the preys. The ultrastructure of the dominant types of hemocytes, such as granulocytes, plasmatocytes and prohemocytes, was evaluated using transmission electron microscope (TEM). Quantitative evaluation of apoptotic and necrotic cells, as well as the ones with depolarized mitochondria in hemolymph, was performed using flow cytometry, while ATP concentration and ADP/ATP ratio in hemocytes were measured by luminescent methods. Cadmium, unlike copper, demonstrated proapoptotic and pronecrotic activity. Low ATP levels and high ADP/ATP ratio in hemocytes indicate a disturbance in the energy metabolism of cells and may account for their qualitative and quantitative degenerative changes. The intensification of death processes in hemocytes after an exposure to cadmium-contaminated food may impair the ability of these cells to fight infectious diseases. Copper at the applied dosage was safe for the spiders without causing visible changes in the hemocyte ultrastructure and in the level of analyzed cell death indices.

Studies on acute and chronic toxicity of cadmium to freshwater snail Lymnaea acuminata (Lamarck) with special reference to behavioral and hematological changes.

Molluscs have long been regarded as promising bioindicator and biomonitoring subjects for heavy metals as molluscs are highly tolerant to heavy metals and exhibit high accumulation in their body. In spite of several previous studies about the impact of cadmium on molluscs, little information exists in literatures concerning the toxic effects of cadmium on Lymnaea acuminata, especially pertaining to behavioral and hematological changes as these are considered effective bioindicators and biomonitoring variables for detecting heavy metals in polluted water bodies. In the present study, the median lethal concentrations of cadmium chloride to snail, Lymnaea acuminata, were estimated to be 9.66, 7.69, 6.26, and 5.54 mg/L at 24, 48, 72, and 96 h, respectively. For behavioral studies, variable test concentrations of cadmium from 0.00 to 10 mg/L were used. The clumping tendency, crawling activity, and touch reflex in the exposed snails were gradually decreased with higher concentrations at 72 and 96 h. For measuring the hemocyte numbers in the circulating hemolymph of snail during chronic cadmium exposure, two sublethal doses of cadmium (10 and 20% 96-h LC50-0.55 and 1.11 mg/L, respectively) were used. A significant variation (p < 0.05) from the control at all exposure times (7, 14, 21, and 28 days) was recorded at 1.11 mg/L concentration. The total count of circulating hemocytes was significantly reduced (p < 0.05) compared to the controls at both concentrations of cadmium exposure at all time periods except 14 and 21 days exposure at 0.55 mg/L where values were non-significantly increased. In comparison between two sublethal doses, blood cells were significantly (p < 0.05) lowered at 1.11 mg/L cadmium treatment. Considering the behavioral and hematological data, it seems possible to forecast the physiological state of snails in cadmium-contaminated water bodies and these findings can be used in determining the safe disposal level of cadmium in aquatic ecosystem.

Sub-lethal effects of waterborne exposure to copper nanoparticles compared to copper sulphate on the shore crab (Carcinus maenas).

The toxicity of soluble copper (Cu) to marine organisms is reasonably well described. However, the hazard of Cu engineered nanomaterial (ENMs) is poorly understood. The aim of the present study was to compare the toxicity of Cu ENMs to Cu as CuSO4 in the shore crab, Carcinus maenas. The crabs were exposed via the water using a semi-static approach to 0.2 or 1mgL-1 of Cu ENMs or 1mgL-1 of Cu as CuSO4. Gills, hepatopancreas, chela muscle and haemolymph were collected at days 0, 4 and 7 for the body burden of Cu, histology and biochemical analysis [thiobarbituric acid reactive substances (TBARS) and total glutathione (GSH)]. Nominal exposure concentrations of both the ENMs and the metal salt were maintained at over 80% in each treatment throughout the experiment. By day 7, 54% mortality was recorded in the 1mgL-1 CuSO4 treatment, compared to just 21% in the 1mgL-1 Cu ENM-exposed crabs. The target organs for Cu accumulation were similar for both forms of Cu with highest concentrations in the gills, particularly the posterior gills; followed by the hepatopancreas, and with the lowest concentrations in the chela muscle. No changes were observed in the osmolarity of the haemolymph (ANOVA, P>0.05). TBARS were measured as an indicator of lipid peroxidation and showed the greatest change in the anterior and posterior gills and hepatopancreas of animals exposed to 1mgL-1 Cu ENMs (ANOVA or Kruskal-Wallis, P<0.05). No statistically significant changes in total GSH were observed (ANOVA, P>0.05; n=6 crabs per treatment). Histological analysis revealed organ injuries in all treatments. The types of pathologies observed in the Cu ENM treatments were broadly similar to those of the Cu as CuSO4 treatment. Overall, the target organs and Cu accumulation from Cu ENMs were comparable to that following exposure to Cu as CuSO4, and although there were some differences in the sub-lethal effects, the metal salt was more acutely toxic.

Effects of waterborne copper delivered under two different exposure and salinity regimes on osmotic and ionic regulation in the mudflat fiddler crab, Minuca rapax (Ocypodidae, Brachyura).

The effects of exposure to copper (Cu) on tissue Cu accumulation, on hemolymph osmotic, Na+ and Cl- regulation, and on gill Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) activities were evaluated in the fiddler crab Minuca rapax. Waterborne copper was delivered to the crabs at one of three salinities (seawater at 25‰ salinity [S] = isosmotic control; distilled water [<0.1‰ S] = hypo-osmotic medium; or 60‰ S = hyper-osmotic seawater) either for 5 days in a 0.5-cm water film containing 0, 50, 150, 250 or 500µg Cu/L with free access to a dry surface, or in crabs fully submerged for 5h at 0, 250 or 500µg Cu/L. In the crabs with free access to a dry surface, the highest Cu concentrations were found in the hemolymph and hepatopancreas with some accumulation in the gills; accumulation in the hemolymph and gills was enhanced in low salinity but was salinity independent in the hepatopancreas. Osmotic regulation was unaffected by Cu exposure; however Na+ and Cl- hypo- regulation was impaired by Cu in 25 and 60‰ S. Gill NKA activity was stimulated 2-fold at 50µg Cu/L and markedly inhibited at 150µg Cu/L and above in 0 and 25‰ S. Gill CA was inhibited in <0.1‰ S but stimulated in 25 and 60‰ S; an inverse concentration-CA activity response was seen above 150µg Cu/L for all salinities. In the submerged crabs, Cu accumulated in all tissues in 60‰ S; however, there was no clear-cut Cu concentration-accumulation relationship evident in any tissue for either exposure regime, likely owing to the crabs' ability to regulate Cu. Copper exposure diminished osmotic, [Na+] and [Cl-] hypo-regulatory ability, especially in higher salinities. Gill NKA activity was markedly inhibited by Cu overall, and particularly above 250µg Cu/L in <0.1‰ S. Gill CA activity was inhibited in 25‰ S but inconsistently affected in 0 and 60‰ S. These findings show that Minuca rapax is affected both physiologically and biochemically by Cu contamination, although to different degrees, depending on the delivery regime, salinity, copper concentration and target tissue.

Humoral and cellular immune responses induced by the urease-derived peptide Jaburetox in the model organism Rhodnius prolixus.

BACKGROUND: Although the entomotoxicity of plant ureases has been reported almost 20 years ago, their insecticidal mechanism of action is still not well understood. Jaburetox is a recombinant peptide derived from one of the isoforms of Canavalia ensiformis (Jack Bean) urease that presents biotechnological interest since it is toxic to insects of different orders. Previous studies of our group using the Chagas disease vector and model insect Rhodnius prolixus showed that the treatment with Jack Bean Urease (JBU) led to hemocyte aggregation and hemolymph darkening, among other effects. In this work, we employed cell biology and biochemical approaches to investigate whether Jaburetox would induce not only cellular but also humoral immune responses in this species. RESULTS: The findings indicated that nanomolar doses of Jaburetox triggered cation-dependent, in vitro aggregation of hemocytes of fifth-instar nymphs and adults. The use of specific eicosanoid synthesis inhibitors revealed that the cellular immune response required cyclooxygenase products since indomethacin prevented the Jaburetox-dependent aggregation whereas baicalein and esculetin (inhibitors of the lipoxygenases pathway) did not. Cultured hemocytes incubated with Jaburetox for 24 h showed cytoskeleton disorganization, chromatin condensation and were positive for activated caspase 3, an apoptosis marker, although their phagocytic activity remained unchanged. Finally, in vivo treatments by injection of Jaburetox induced both a cellular response, as observed by hemocyte aggregation, and a humoral response, as seen by the increase of spontaneous phenoloxidase activity, a key enzyme involved in melanization and defense. On the other hand, the humoral response elicited by Jaburetox injections did not lead to an increment of antibacterial or lysozyme activities. Jaburetox injections also impaired the clearance of the pathogenic bacteria Staphylococcus aureus from the hemolymph leading to increased mortality, indicating a possible immunosuppression induced by treatment with the peptide. CONCLUSIONS: In our experimental conditions and as part of its toxic action, Jaburetox activates some responses of the immune system of R. prolixus both in vivo and in vitro, although this induction does not protect the insects against posterior bacterial infections. Taken together, these findings contribute to the general knowledge of insect immunity and shed light on Jaburetox's mechanism of action.

A C-type lectin with an immunoglobulin-like domain promotes phagocytosis of hemocytes in crayfish Procambarus clarkii.

C-type lectins are important immune molecules that participate in host defense response. The present work reports a novel C-type lectin (PcLec3) from the red swamp crayfish Procambarus clarkii. Sequence analysis found that PcLec3 encodes a polypeptide with252 amino acid residues, which contains an immunoglobulin-like domain (IG) and a C-type lectin domain (CTLD) arranged in tandem. Tissue distribution analysis indicated that PcLec3 is enriched expressed in hemocytes and hepatopancreas cells, in which PcLec3 was up-regulated following bacterial challenge by Vibrio anguillarum. Function analysis using recombinant full-length PcLec3, IG, and CTLD proteins revealed that these recombinant proteins had the capacity to bind carbohydrates and bacteria, while IG determined the cell binding activity. However, only full-length PcLec3 promotes the phagocytic activity of hemocytes and subsequent clearance of invasive bacteria. Taken together, these results manifest that PcLec3 acts as a hemocyte adhesion molecule to promote hemocyte phagocytosis against invasive V. anguillarum.

Copper-induced oxidative damage to the prophenoloxidase-activating system in the freshwater crayfish Procambarus clarkii.

Previous studies have demonstrated copper-induced proteins damage in gill and hepatopancreas of the freshwater crayfish Procambarus clarkii, but little information is available about its effects on key component of the innate defense in haemolymph. In the present study, we evaluated the relationship between oxidative carbonylation and prophenoloxidase-activating system (proPO-AS) activity, by exposing P. clarkii to sub-lethal concentrations (1/50, 1/12, 1/6 and 1/3 of the 96 h LC50) Cu(2+) up to 96 h. Six biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), protein carbonyl (PC), malondialdehyde (MDA) and DNA-protein crosslinks (DPCs), and six indicators of immune status, i.e. total hemocyte counts (THCs), differential hemocyte counts (DHCs), hemocyanin (HC), prophenoloxidase (proPO), serine protease (SP) and phenoloxidase (PO), were determined in haemolymph. The results indicated that there was a significant increase (P < 0.05) in the levels of ROS, PC, MDA and DPCs accompanied by markedly decreased (P < 0.05) activities of proPO, SP, PO and HC in a dose and time dependent manner. The significant and positive correlations (P < 0.01) between ROS production and the formation of PC, MDA and DPCs were observed in crayfish at 96 h. There was a significant negative correlation (P < 0.01) between the levels of protein carbonyls and the activities of proPO and SP in hemocyte lysate supernatant and PO and HC in haemolymph. Carbonylated proteins may be recognized not merely as a specific signal in oxidative stress pathways but also as a "non-self" molecule in proPO-AS. In crayfish species, copper-catalyzed protein carbonylation may be one of the main mechanisms for immunity dysfunction in proPO-AS.

Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.

Alterations in the oxidative metabolism of Rhipicephalus (Boophilus) microplus ticks in response to exposure to the insect growth regulator fluazuron / Alterações no metabolismo oxidativo de carrapatos Rhipicephalus (Boophilus) microplus em resposta à exposição ao regulador de crescimento fluazuron

Abstract Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the tick’s fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects.

Resumo Com o objetivo de caracterizar os efeitos não-alvo da ação do fluazuron, foram realizados testes bioquímicos para analisar possíveis alterações no metabolismo de carboidratos em carrapatos Rhipicephalus (Boophilus) microplus após sua exposição ao composto. Foram coletados hemolinfa e corpo gorduroso de fêmeas ingurgitadas antes e após (4, 8 e 15 dias) a exposição ao fluazuron. Análises espectrofotométricas foram usadas para quantificar a concentração de glicose e a atividade da lactato desidrogenase (LDH) na hemolinfa e concentração de glicogênio no corpo gorduroso. Cromatografia Líquida de Alta Eficiência (CLAE) foi usada para determinação das concentrações de ácidos carboxílicos na hemolinfa e avaliar possíveis alterações em metabolismo intermediário em relação ao consumo de oxigênio. Aumento na atividade de LDH e concentração de ácido lático indicaram que o fluazuron pode regular o metabolismo fermentativo em carrapatos. A exposição ao fluazuron também aumentou a concentração de glicose na hemolinfa, apesar de não ter havido diferença significativa na comparação entre as médias no mesmo dia de avaliação. Além de aumentar o conhecimento sobre o modo de ação do fluazuron, investigações sobre tais mecanismos também são úteis no descobrimento de novos efeitos secundários ainda não explorados.